Project Summary: Characterizing global regulatory networks in human embryonic stem cells Human embryonic stem cells (hESCs) provide a model for early preimplantation development and have the potential to be harnessed for applications in regenerative medicine. However, a comprehensive, integrated view of the regulatory network underlying hESC identity is fundamentally lacking. The overarching goal of this proposal is to elucidate novel features of the global regulatory architecture in hESCs. In order to discover critical components of this network, a genome-wide shRNA screen was performed, identifying a number of previously unrecognized transcription factors and epigenetic modifiers essential to hESC biology, such as EP400, an epigenetic modifier that notably acts as a transcriptional activator in other cell types. Screen hits were characterized and narrowed to a final shortlist of 15 novel genes with a clear role in hESC maintenance. These factors will be sorted into functional categories and organized into wider, integrated clusters of co- regulation within the hESC network. The first aim will seek to characterize this newly defined set of novel, screen-identified factors alongside known, established hESC factors. Loss-of-function experiments will be used to sort each gene into functional categories defined by knockdown effects on pluripotency, self-renewal, or viability. Further characterization of gene function in hESCs will be conducted using functional assays and analysis of molecular markers. The second aim will employ global deep-sequencing-based approaches to identify relationships between critical hESC factors and reconstruct co-regulatory modules important to the maintenance of hESCs. Transcription factors will be clustered by their impact on global expression profile upon knockdown, with patterns of expression changes shared by co-regulators. Binding site analysis will allow for specific mapping of the regulatory circuitry that establishes and maintains the hESC transcriptional landscape. Finally, the same profiling methods will be used to integrate EP400 and its associated histone variant H2A.Z into this network to determine whether the structure of this epigenetic pathway aligns with downstream transcriptional modules as hypothesized, or displays its own unique, independent organization. The proposed studies will thus provide a systems view of the mechanisms by which transcriptional and epigenetic regulators converge to drive the unique programs of pluripotency and self-renewal in the early human embryo. A better understanding of the complex, network-based regulation governing hESC behavior will help address the need for more accessible models of human development and guide the advancement of regenerative therapies such as somatic cell reprogramming.